This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Salmonella spp. and oxyurids are among the most prevalent bacterial and parasitic agents in
reptiles. These organisms are routinely isolated in healthy tortoises, although heavy
infections may cause significant pathology. Tortoises are considered a common source
of reptile-associated salmonellosis, an important zoonosis reported worldwide. A survey
of the prevalence of Salmonella spp. and oxyurids in 53 tortoises was conducted in southern Italy and a possible
correlation between the two pathogens was therefore investigated.

Results

Salmonella spp. and oxyurids were detected with a prevalence of 49.1 and 81.1%, respectively.
A significant positive correlation between Salmonella spp. and oxyurids was demonstrated. However, confounding factors related to husbandry
could have been involved in determining this correlation.

Conclusions

Our results suggest that caution should be exercised in translocation, husbandry,
and human contact with tortoises and other exotic pets. Further studies on the epidemiology,
molecular characterization and pathogenesis of Salmonella and oxyurids are needed to assess the actual impact of these organisms, as single
or associated infections, on tortoises and on other exotic pets.

Background

It is estimated that about 640 000 live reptiles are traded annually worldwide [1]. Although some species of tortoises are listed on Appendix II of the Convention on
International Trade in Endangered Species (CITES) and thus subjected to strict trade
regulations [2], there is a high demand for these reptiles to be kept as pets. Since the enactment
of regulations implementing the CITES convention in the European Union (EU), several
applications have been submitted for the CITES registration of privately owned tortoises
in Italy; however, there are no official figures on the number of tortoises raised
as pets in Italy [3]. The illegal introduction of reptiles raises public health concerns because these
animals can be infected by various pathogens (virus, bacteria, protozoa, helminthes
and arthropoda) and some of them are zoonotic [4].

Among bacteria, Salmonella spp. [5,6] is frequently reported in tortoises [6-8]. It is considered to be part of the intestinal flora and does not cause significant
clinical sign, except in stressed or immunocompromised animals, where it may cause
salmonellosis, an important reptilian zoonosis [8,9].

Several species of oxyurids commonly inhabit the colon of tortoises but are rarely
considered pathogenic or zoonotic [10,11]. Heavy infections might be one cause of anorexia in tortoises coming out of hibernation
[12]. Studies conducted so far in Italy showed oxyurids as the most prevalent nematode
parasites in these animals [13-15].

We investigated the prevalence of Salmonella spp. and oxyurids in tortoises farmed in southern Italy. A secondary objective was
to assess a possible correlation between the two pathogens investigated.

Methods

Ethics statement

Tortoises handling procedures were performed with the license of the farmers. The
animals used in the present study were sampled following approval by the animal ethics
and welfare committee of the University of Naples Federico II (in Italian, Comitato
Etico-scientifico per la Sperimentazione Animale dell’ Università di Napoli Federico
II; protocol number 0021099).

Animals and sampling

Between May and August 2011, faecal samples were collected from 53 clinically healthy
adult tortoises belonging to five species as indicated in Table 1. In particular, the species of tortoises examined were separated by species and were
represented by: Testudo hermanni (22 T. hermanni ssp. hermanni and 8 T. hermanni ssp. boettgeri), Testudo marginata (12 T. marginata ssp. marginata, and 1 T. marginata ssp. weissingeri), Testudo graeca (7), Testudo horsfieldii (2), and Geochelone carbonaria (1).

Table 1.Prevalence of Salmonella and oxyurids in the different species of tortoises farmed in southern Italy

45 female and 7 male tortoises were examined and were selected from six private farms
located in urban areas in the Campania region of southern Italy. Tortoises were housed
in terrarium systems.

In order to perform bacteriological and parasitological analyses, each tortoise was
lying carefully in a tank and one cloacal swab sample and one faecal sample for each
reptile were collected. Cloacal swab samples were inoculated in Buffered Peptone Water
(Oxoid, Milan, Italy), whereas faecal samples were preserved in formalin 5% and then
stored at +4°C until further analyses.

Bacteriological analysis

Cloacal swab samples were incubated at 37°C for 18 h. After incubation, samples were
inoculated into Rappaport–Vassiliadis Broth (Oxoid) and incubated at 42°C for 18 h.
Cultures obtained were plated onto Xylose-Lysine-Desoxycholate Agar (Oxoid), incubated
at 37°C and examined after 24 h. Suspected Salmonella colonies were inoculated onto a second selective media, Brilliant Green Agar (Oxoid)
and incubated at 37°C for 24 h. All isolates were biochemically identified by using
the API20-E system (bioMérieux, Milan, Italy). All strains were stored frozen at –80°C
in 20% glycerol until serotyping was performed. Salmonella isolates were serotyped according to the Kauffman-White scheme. Analyses were carried
out in collaboration with the National Reference Laboratory for Salmonella (IZSVe, Legnaro, Italy).

Parasitological analysis

Parasitological analyses were performed using the FLOTAC Pellet Technique [16]. This technique is performed for samples with an unknown weight of faecal material.
In these circumstances, the weight of the faecal material to be analyzed can be inferred
by weighing the sediment in the tube (pellet) after filtration and centrifugation
of the faecal sample [17]. Formalin 5% was added to each faecal sample to reach a final volume of 20 ml; each
sample was homogenized and filtered. Two 15 ml conical tubes were filled with the
filtered suspension up to 6 ml and were centrifuged for 3 min at 1,500 rpm. After
centrifugation the supernatant was discarded and the two pellets (sediments) were
weighed. Two different flotation solutions were used to re-suspend the pellets: FS2
(Sodium Chloride Solution) (1200 s.g.) and FS7 (Zinc Sulphate Solution) (1350 s.g.).
After homogenization, each of the two suspensions was poured into the two flotation
chambers of the FLOTAC apparatus. The FLOTAC was closed and centrifuged for 5 min
at 1,000 rpm; after centrifugation, the top parts of the flotation chambers were translated
and each chamber was read under the microscope.

Parasitic elements (eggs, larvae, oocysts and cysts of oxyurids) were counted, photographed
and measured using a light microscope at 10X and 40X magnifications (Leica DFC 490)
and identified in accordance with Schneller and Pantchev [18].

Statistical analysis

For each tortoise, oxyurid eggs per gram (EPG) of faeces was calculated using the
following formula: EPG = (N x 1.2)/wp where N is the number of eggs counted and wp
is the weight of the pellet. Therefore, the mean EPG (for positive individuals) was
calculated per each tortoise species. The relationship between Salmonella positivity and oxyurid EPG was evaluated by utilizing the Spearman’s Rho correlation performed using SPSS software (Version 13).

With respect to parasitological results, eggs of oxyurids were recorded in 43 out
of the 53 tortoises examined (81.1%; 95% CI = 67.6 – 90.1%). Among the positive individuals,
oxyurid EPG values ranged from 5 to 31,187. Table 1 indicates the mean oxyurid EPG for each tortoise species. Out of the 53 tortoises
examined 23 (43.4%) were affected with both Salmonella spp. and oxyurids. The statistical analysis showed a significant positive correlation
between oxyurid EPG and Salmonella (Spearman’s Rho = 0.499; P = 0.000).

The only Geochelone carbonaria tested was consistently negative both for Salmonella spp. and oxyurids. Other parasitic elements were also found in all the examined tortoises
using the FLOTAC technique, indicating infection by ascarids, Entamoeba spp., Nyctotherus and Balantidium coli.

Discussion

The findings of the present study showed a prevalence of 49.1% for Salmonella spp. and 81.1% for oxyurids in tortoises farmed in southern Italy. Previous studies
on the prevalence of Salmonella in tortoises conducted worldwide show heterogeneous results. Recovery rates of Salmonella in the present study are similar to the data reported by Savage and Baker [19] and Percipalle et al. [3] who reported a prevalence of 38.0% and 34.1%, respectively. Nevertheless, a recent
survey conducted by Nowakiewicz et al. [20] showed levels of infection lower than those reported in the present study with a
prevalence of 18.7%, whereas a study conducted by Pasmans et al. [7] showed levels of infection higher than those reported in our present study with a
prevalence of 79.0%. The reasons for this recovery rate variation are not clear. The
methods of isolation, species of tortoise, and geographical circumstances could have
been involved in explaining these variable results.

The most prevalent Salmonella serotypes isolated in the present study were S. Richmond, S. Abony, and S. Hermannswerder which have been previously isolated in both captive and free-living
tortoises [3,7,21]. It is noteworthy that some of the serotypes isolated during the present study have
been associated with outbreaks of human salmonellosis worldwide. S. Kottbus have been associated with an outbreak in infants in Spain linked to contamination
of commercial bottled water [22]. Another study conducted by Ryder et al. [23] suggests that S. Kottbus may colonize the human mammary gland and thus be transmitted to infants
by breastfeeding. S. Richmond has been isolated during an outbreak of salmonellosis in a military detachment
in Spain [24] and during an outbreak of dysentery in children in Malaysia [25].

It is not possible to speculate regarding the source of Salmonella infections in the present study because they may have been initiated before or after
introduction of tortoises in the farms. The higher prevalence of certain serotypes
compared to others could be a result of the potential cross-infection between animals
which occurred during housing in the farms.

With respect to the parasitological analysis our results showed a prevalence of 81.1%
for oxyurids; higher values compared with those recently reported in a study conducted
by Papini et al. [15] who recorded a prevalence of 29.2% for these pinworms in captive tortoises farmed
in central Italy. We can explain this difference in the prevalence rates by the different
diagnostic tools used in the two studies. Papini et al. [15] used a routine faecal flotation method to detect helminthes. In contrast, we used
the highly sensitive and multivalent FLOTAC technique which was also showed as the
best copromicroscopic method for assessing pinworm prevalence as reported by Rinaldi
et al. [26] in rabbits.

Interestingly, a significant positive correlation between Salmonella spp. and oxyurid EPG was observed in our study. Tortoises with high oxyurid EPG values
were also positive to Salmonella spp. It is well demonstrated that parasites may be affected, directly or indirectly,
by cytokines and other immune effector molecules and parasites may themselves produce
factors that affect the cells of the immune system [27]. The consequent immunomodulation may enhance host susceptibility to other infectious
pathogens [27] such as Salmonella spp. Furthermore, the interactions between parasites and other infectious agents
have been described in various surveys in literature. Cringoli et al. [28] reported the co-presence of antibodies to Neospora caninum and Leishmania infantum in dogs suggesting that one protozoan may enhance the susceptibility to other one.
Rinaldi et al. [29] showed N. caninum and Bovine herpesvirus 1 (BHV-1) coinfection in cattle, demonstrating the role of
N. caninum as a primary pathogen and its presence as a risk factor for BHV-1 infection in this
animal species. These studies, in line with the present study, suggest that in mixed
infections the burden of one or both the infectious agents may be increased, one or
both may be suppressed or one may be increased and the other suppressed. However,
the correlation found between Salmonella and oxyurids in the present study does not necessarily reflect a causal relationship
between the two organisms. Indeed confounding factors related to husbandry could have
been involved in determining this correlation. It is very likely that this correlation
might be also enhanced by the habitat (sanitation, temperature, diet) of the tortoises
and further studies are needed to address this issue.

Conclusions

In conclusion, the findings of the present study showed a significant positive correlation
(Spearman’s Rho = 0.499; P = 0.000) between Salmonella and oxyurid infection in tortoises farmed in southern Italy. Further studies on the
epidemiology, molecular characterization and pathogenesis of Salmonella and oxyurids, as well as on the role of husbandry in the correlation by the two pathogens,
are needed to assess the actual impact of these organisms, as single or associated
infections, on tortoises and other exotic pets. In addition, as direct transmission
of pathogens (as Salmonella) to humans may occur through handling of an infected reptile and indirect transmission
may occur through contact with feces or by handling objects contaminated by reptiles,
results presented here suggest that those individuals who handle tortoises should
emphasize sanitary precautions.

Consent

Consent was obtained from the owner of the animal for publication of this study.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

LD and LR conceived the study, designed the experiments, analyzed the experimental
data and prepared the manuscript. AF and GC2 supervised the experiments and helped
draft manuscript. MPM and PP performed the parasitological analysis. TPR performed
the bacteriological analysis. MC and GC1 performed the sampling. All authors read
and approved the final manuscript.

Acknowledgements

The authors thank Dr. Antonia Ricci, Dr. Lisa Barco and other personnel of the National
Reference Laboratory for Salmonella (IZSVe, Legnaro, Italy) for serotyping of Salmonella strains.